Micropeptide AF127577.4-ORF hidden in a lncRNA diminishes glioblastoma cell proliferation via the modulation of ERK2/METTL3 interaction

Micropeptides hidden in long non-coding RNAs (lncRNAs) have been uncovered to program various cell-biological changes associated with malignant transformation-glioblastoma (GBM) cascade. Here, we identified and characterized a novel hidden micropeptide implicated in GBM. We screened potential candidate lncRNAs by establishing a workflow involving ribosome-bound lncRNAs, publicly available MS/MS data, and prognosis-related lncRNAs. Micropeptide expression was detected by western blot (WB), immunofluorescence (IF), and immunohistochemistry (IHC). Cell proliferation rate was assessed by calcein/PI staining and EdU assay. Proteins interacted with the micropeptide were analyzed by proteomics after co-immunoprecipitation (Co-IP). We discovered that lncRNA AF127577.4 indeed encoded an endogenous micropeptide, named AF127577.4-ORF. AF127577.4-ORF was associated with GBM clinical grade. In vitro, AF127577.4-ORF could suppress GBM cell proliferation. Moreover, AF127577.4-ORF reduced m6A methylation level of GBM cells. Mechanistically, AF127577.4-ORF diminished ERK2 interaction with m6A reader methyltransferase like 3 (METTL3) and downregulated phosphorylated ERK (p-ERK) level. The ERK inhibitor reduced p-ERK level and downregulated METTL3 protein expression. AF127577.4-ORF weakened the stability of METTL3 protein by ERK. Also, AF127577.4-ORF suppressed GBM cell proliferation via METTL3. Our study identifies a novel micropeptide AF127577.4-ORF hidden in a lncRNA, with a potent anti-proliferating function in GBM by diminishing METTL3 protein stability by reducing the ERK2/METTL3 interaction. This micropeptide may be beneficial for development of therapeutic strategies against GBM.

As an extremely fatal malignancy, glioblastoma (GBM) has become one of the most challenging cancers due to its highly refractory to therapies (chemoradiotherapy and immunotherapy) and grim prognosis (2-year survival of about 25%) 1,2 .Despite advanced knowledge in molecular etiology and pathology and the clinical trials in multiple experimental drugs, the patients of GBM remain unprofited for recent years 1,3 .
In order to develop innovatively efficient treatments against GBM, numerous studies have focused on investigating epigenetic modulators that participate in GBM malignant process 4,5 .Among these modulators, long noncoding RNAs (lncRNAs), previously annotated as transcripts lacking protein-coding capacity, have been proved to program a variety of cell-biological alterations associated with malignant transformation-GBM cascade 6,7 .Lately, with improved sequencing methodologies, some lncRNAs have been revealed to harbor short open reading frames (ORFs) (usually < 300 nucleotides) that possess the ability to encode hidden micropeptides 8,9 .These hidden micropeptides have modulatory activities that are implicated in a variety of cellular processes 10 .More www.nature.com/scientificreports/intriguingly, these hidden micropeptides can serve as new cancer biomarkers, participators, and therapeutic targets 11,12 .
Here, we report that the ORF (location of lncRNA: 1483-1569 bp) of AF127577.4,which was previously baptized as a lncRNA, can generate a 29-aa endogenous micropeptide AF127577.4-ORF.AF127577.4-ORFfunctions as a suppressor of GBM cell proliferation in vitro by diminishing the stability of m6A writer methyltransferase like 3 (METTL3), a pro-tumorigenic player in GBM by elevating RNA m6A methylation 13,14 , via its modulation of the ERK2/METTL3 interaction.The novel micropeptide hidden in lncRNA AF127577.4may be beneficial for development of therapeutic strategies in GBM.

Prediction of the coding potential of GBM-related lncRNAs
The hidden polypeptides generated by lncRNAs have emerged as critical players in tumor biology 15 .As a result, we here sought to identify potential micropeptides hidden in GBM-associated lncRNAs.To address this, we downloaded Ribo-seq dataset GSE129757 of LN308 GBM cells from GEO database and obtained 3607 ribosomebound lncRNAs (count > 0) after ensembol search tool for sequence acquirement.We then searched these presumable lncRNAs for hypothetical ORFs using EMBOSS getORF tool, excluded the sequences with less than 15 nucleotides, and ended up with 46,678 hypothetical ORFs and their respective aa sequences, which we called the generated hypothetical lncRNA-encoded micropeptide FASTA database.Meantime, the proteomics analysis raw files (PXD013541) of LN308 GBM cells, which were based on the MS/MS, were obtained from the database PRoteomics IDEntifications.The obtained raw files were subsequently processed by MaxQuant software using the generated hypothetical micropeptide FASTA database.From this, we discovered 84 hypothetical micropeptides (Supplementary Table S1) hidden in 32 lncRNAs (Supplementary Table S2).
To further identify GBM prognosis-related micropeptides hidden in lncRNAs, we then retrieved lncRNAs that were related to poor prognosis of GBM patients from GBM RNA-seq data from TCGA database.A total of 5269 lncRNAs was identified by using R survival package (Supplementary Table S3).Next, we combined the obtained 32 lncRNAs with coding potential and 5269 lncRNAs associated with GBM poor prognosis and discovered 17 common lncRNAs (Supplementary Table S4), as illustrated by Venn diagram (Fig. 1A).Based on the inclusion criteria that the optimal length of micropeptide is generally 25-100 aa, a total of 7 lncRNAs were screened (Supplementary Table S5).Among these transcripts, we found that 3 prognosis-related lncRNAs AC090114.2,AL158166.2and AF127577.4(Fig. 1B) that were aberrantly expressed in GBM tumors and had close relevance to overall survival (OS), IDH status, WHO grade and histological type (Fig. 1C).

AF127577.4-ORF suppresses proliferation of GBM cells
Given the AF127577.4-ORFassociation with GBM pathological grade, we then asked if the micropeptide could affect GBM cell growth.For these experiments, we elevated the expression of AF127577.4-ORFby the expression construct in LN229 and U251 GBM cells.AF127577.4-ORFincrease dramatically induced cell death as measured by the ratio of PI (red)/Calcein (green)-stained cells (Fig. 4A).EdU analysis presented the reduction of the EdU-positive cells after AF127577.4-ORFincrease (Fig. 4B), which suggested that AF127577.4-ORFcould diminished GBM cell proliferation.We also examined the expression change of Ki67 and PCNA, two proliferating markers, in AF127577.4-ORF-transfectedGBM cells.Via IF microscopy, the protein levels of Ki67 and PCNA were strongly downregulated by AF127577.4-ORF(Fig. 4C,D).

Identification of downstream molecular process of AF127577.4-ORF
We next investigated molecular determinants by which AF127577.4-ORFparticipates in GBM process.To address this, we elevated AF127577.4-ORFexpression in LN229 GBM cells via the Flag fusion expression construct and carried out Co-IP experiment by using rabbit polyclonal antibody against Flag tag.Silver staining technique after SDS-PAGE revealed a specific band in Flag-containing immunoprecipitates versus isotype anti-IgG controls (Fig. 5A).The subsequent technique of HPLC/MS showed 473 unique proteins coprecipitated by AF127577.4-ORFfused with Flag tag after the exclusion of miscellaneous protein (Fig. 5B and Supplementary Table S6).
We then analyzed the potential function of the 473 proteins and their enrichment signaling pathways.Via the enrichment analysis of GO and KEGG pathways, we discovered that these proteins had a tight relationship with spliceosome, RNA transport, RNA splicing, and RNA helicase activity (Fig. 5C).In addition, String tool (https:// cn.string-db.org/) showed that these proteins could form a complex regulatory network (Supplementary Fig. S1).

AF127577.4-ORF diminishes METTL3 stability via the repression of ERK2/METTL3 interaction
Among the 406 proteins interacted with AF127577.4-ORF,we found multiple m6A methylation-related proteins.We therefore examined if the micropeptide could affect m6A RNA methylation level of GBM cells.Indeed, we observed a reduction in m6A methylation level in LN229 and U251 cells when introduced with AF127577.4-ORFexpression plasmid (Fig. 6A).

Discussion
By establishing the identification workflow of GBM prognosis-related micropeptides hidden in lncRNAs, we found 3 lncRNAs, which were further confirmed to have the ability to encode a micropeptide.Of them, AF127577.4-ORFwas identified as the most significant suppressor of GBM cell viability.Our data also indicated the endogenous expression of AF127577.4-ORF in GBM cells.It is interesting to note that the micropeptide can function as an anti-proliferating protein in GBM cells.The alteration of GBM cell proliferation involves Ki67 17 , which is a well-known marker of proliferation 18 , and PCNA 17 , which is a modulator of DNA synthesis 19 .By determining their expression, we further reinforced AF127577.4-ORF'santi-proliferative role.Furthermore, IHC analysis of clinical specimens suggested the negative relationship between the micropeptide and GBM clinical grade.It's worth noting that AF127577.4lncRNA, which encodes the AF127577.4-ORF,significantly associates with higher GBM grade and reduced patient survival, whilst the AF127577.4-ORFmicropeptide encoded by the same lncRNA is negatively correlated to GBM grade and reduces both cell proliferation and ERK activation and also increases cell death.LncRNAs have been reported to exert their functions through serving as RNA molecules or encoded-peptides/proteins, and the roles of lncRNAs and encoded-peptides/proteins might be entirely different.For instance, Ji et al. reported that LINC00665 contributed to breast cancer progression through miR-379-5p/ LIN28B axis 20 .Also, Guo et al. reported that micropeptide CIP2A-BP encoded by LINC00665 blocked breast cancer progression 21 .This phenomenon may be related to the independent working mechanisms of lncRNA KDM4A-AS1 and KDM4A-AS1-encoded peptide.In addition, AF127577.4-ORF is a micropeptide encoded by lncRNA AF127577.4,but whether there are other open reading frames of this lncRNA with coding ability remains still unclear.In a recent report, Zhang and colleagues uncovered an 87-aa uncharacterized micropeptide hidden in the circular form of lncRNA LINC-PINT, which hinders GBM cell proliferation by downregulating several oncogenes by its interaction with PAF1c 22 .m6A methylation, a uniquely crucial way of RNA modification, is capable of influencing the complexity of tumor progression via the modulation of cancer-related biological functions 23 .In light of the m6A research, emerging interests in developing specific anti-tumor therapeutics have focused on inhibitors of m6A writers or erasers 24 .In the further survey of molecular determinants underlying the anti-proliferating role of AF127577.4-ORFusing HPLC/MS after Co-IP, we found multiple proteins responsible for m6A methylation.Surprisingly, we uncovered the downregulated effect of AF127577.4-ORFon m6A RNA methylation level of GBM cells.Among these m6A-associated proteins, METTL3 was interesting in our current work due to its role as a m6A writer 25 and a pro-tumorigenic player in GBM 13,14 .The activation of the ERK pathway has promoting functions in m6A RNA methylation, thereby contributing to GBM tumorigenesis 26 .Furthermore, through physical interaction, ERK stabilizes METTL3 by phosphorylating METTL3 to upregulate and activate METTL3 and thus promotes m6A RNA methylation 16,27 .Through protein-protein interaction (PPI) analysis, we also found the direct relationship of ERK2 and METTL3, both of which were demonstrated to interact with AF127577.4-ORF in GBM cells.Using the analysis of protein-protein docking poses, we predicted a theoretical AF127577.4-ORF/ERK2/METTL3complex.Our data also pointed out the interference effect of AF127577.4-ORFon ERK2 interaction with METTL3.Activated ERK can directly phosphorylate METTL3 at three residues and enhance METTL3 activity 16,27 .Our findings showed that ERK inactivation can diminish METTL3 protein level.Because AF127577.4-ORFdid not www.nature.com/scientificreports/affect METTL3 mRNA expression, we first established evidence that AF127577.4-ORFdiminishes METTL3 protein stability by suppressing ERK2/METTL3 interaction and inactivating ERK.Additionally, via rescue experiments, we demonstrated the anti-proliferating function of AF127577.4-ORF in GBM cells through METTL3.The regulation of AF127577.4-ORF in METTL3 phosphorylation via ERK will be analyzed in GBM cells in the future.
In GBM, upregulated METTL3 can enhance m6A methylation of ADAR1 mRNA and upregulate its protein level, thereby inducing the occurrence of pro-tumorigenic events 13 .In GBM microvascular endothelial cells, METTL3 promotes CPEB2 mRNA stability through the induction of m6A mRNA methylation and thus regulates the permeability of blood-tumor barrier 28 .The involvement of these target genes of METTL3 in the regulation of the AF127577.4-ORF/ERK2/METTL3cascade will be deeply explored in the next step.Although we have revealed the association of AF127577.4-ORFlevel with GBM clinical grade, the precise function of the micropeptide in tumorigenesis and GBM progression remains to be defined using various in vivo experimental models.
Overall, our findings shed light on a novel micropeptide AF127577.4-ORF,which is generated by lncRNA AF127577.4,with a potent anti-proliferating function in GBM by diminishing the protein stability of m6A writer METTL3 by the suppression of the ERK2/METTL3 interaction.The micropeptides hidden in lncRNAs may provide insight for GBM development and could have potential therapeutic values in GBM.

Data retrieving, processing tool, and bioinformatics analysis
The data of ribosome-bound lncRNAs were retrieved from Ribo-seq dataset GSE129757 of LN308 GBM cells from GEO database.We searched their sequences using publicly available ensembol tool (http:// asia.ensem bl.org/ index.html).The hypothetical ORFs of these presumable lncRNAs were predicted by EMBOSS getORF tool (http:// emboss.bioin forma tics.nl/ cgi-bin/ emboss/ getorf).The mass spectrometry (MS)/MS proteomics analysis raw files (PXD013541) of LN308 GBM cells were downloaded from online database PRoteomics IDEntifications 29 and processed by MaxQuant software (http:// www.maxqu ant.org).TCGA database containing GBM RNA-seq data was mined to retrieve the lncRNAs related to poor prognosis of GBM patients and the data were processed by R survival package.We assessed the protein-protein interaction using online tool String (https:// cn.stringdb.org/).Analyses of GO and KEGG pathway enrichment were executed by available database DAVID 30 .We observed the protein-protein docking pose using available tools ZDOCK 3.0.2 31, Prodigy (https:// wenmr.scien ce.uu.nl/ prodi gy/) for binding energy evaluation, and PyMOL 2.5.4 for visualization.
For transfection of GBM cell lines, we applied Lipofectamine 3000 (Invitrogen, Thermo Fisher Scientific) as described elsewhere 32 .In brief, GBM cells were plated at a suitable density to yield approximately 70% confluence at the time of transient transfection (usually at 24 h post-seeding).Lipofectamine reagent and the indicated plasmid(s) were mixed in Opti-MEM media, and followed by addition to each well.Six hours later, fresh regular media were employed to culture cells for 48 h.

Clinical specimens and immunohistochemistry (IHC) for AF127577.4-ORF
Collection and use of clinical specimens were authorized by Xinxiang Central hospital Ethic Committee.All procedures were performed in accordance with Declaration of Helsinki.All subjects signed informed consent in writing before collection of 20 fresh biopsies samples (3 normal glial tissues, 7 clinical pathological I + II grade tumors, and 10 cases III + IV grade tumors) from 17 GBM patients from our hospital.These specimens were selected in accordance with a clear pathological diagnosis.

Calcein/PI staining
After the relevant transfection, Ln229 and U251 GBM cells were tested for cell death rate using Calcein/PI staining based on a double staining kit (#G1707, Servicebio).The death rate was scored by calculating the ratio of red/green cells.

Proliferation analysis
The measurement of the EdU-positive cells was used for evaluation of the proliferation ability of transfected GBM cells.The Click-iT EdU-594 proliferation detection kit (#G1603, Servicebio) was applied for this assay as suggested.Transfected GBM cells were processed by 10 μM of EdU reagent for 2 h, and followed by fixation and permeabilization.Then, the working reagent containing iF594 fluorochrome (red) was added in each well.Following the 30-min incubation, cells were washed, and Hoechst 33,342 staining (blue) was done.

Co-immunoprecipitation (Co-IP), silver staining and MS
For analysis of proteins interacted with AF127577.4-ORF,total extractions of LN229 GBM cells expressing AF127577.4-ORF-Flagpeptide were processed by Co-IP using anti-Flag affinity Sepharose 4B gels (#20585ES01, Yeasen, Shanghai, China).The coprecipitated proteins were separated prior to SDS-PAGE, and the gels were stained with silver using a staining kit and accompanying protocols (#G2080, Servicebio).The extracted protein mixtures were subjected to HPLC-MS by Qinglianbio Biotechnology (Beijing, China) using L-3000 HPLC System.The RAW files were processed by homo sapiens database using Proteome Discoverer2.4software.

Measurement of m6A level
To assess the impact of AF127577.4-ORF in m6A methylation level, we utilized the m6A RNA methylation assay kit and protocols (#ab233491, Abcam).Briefly, in a 96-well dish, 80 μl binding solution was used before addition of 200 ng of RNA extracted from vector-or AF127577.4-ORFplasmid-introduced GBM cells, which was followed

Figure 5 .
Figure 5. Interacted proteins of AF127577.4-ORF in LN229 cells.(A) Silver staining technique after SDS-PAGE of total extractions (Input), proteins pulled down by anti-Flag tag antibody or isotype anti-IgG control.(B) Venn diagram displaying the number of the proteins pulled down by anti-Flag tag antibody or isotype anti-IgG control after HPLC/MS.(C) The bubble plot revealing the closely associated biological processes and significantly enriched KEGG pathways of the unique proteins pulled down by micropeptide AF127577.4-ORF.

Figure 6 .
Figure 6.AF127577.4-ORFweakens METTL3 stability via reducing ERK2/METTL3 interaction.(A) The mRNA m6A methylation level of LN229 and U251 GBM cells after introduction by AF127577.4-ORFplasmid or vector control.(B) IB of Flag-containing immunoprecipitates and IgG controls after Co-IP assays and total cellular lysates (Input) showing the enrichment levels of ERK2 and METTL3.(C) Schematic of the predicted AF127577.4-ORF/ERK2/METTL3complex analyzed using the protein-protein docking poses.(D) Co-IP experiments were performed in AF127577.4-ORFplasmid-or control vector-transfected LN229 cells using antibodies against METTL3 or ERK2.Then, the enrichment level of ERK2 or METTL3 was gauged by IB analysis.(E) Detection of p-ERK and ERK2 levels in GBM cells transfected as indicated by IB analysis.(F) GBM cells were treated with or without SCH772984 and followed by detection of p-ERK, ERK2, and METTL3 levels by IB. (G) Real-time qPCR of GBM cells transfected as indicated detected METTL3 mRNA level.H, LN229 and U251 GBM cells transfected as indicated were pre-treated with or without LM22B-10 and then incubated with cycloheximide (CHX) for 0, 2 and 4 h, and followed by detection of METTL3 protein level.ns: no significant.